The aim of this work is to study the influence of the Physicochemical properties of the lipid bylayer of the membrane associated Na,K-ATPase. A series of fluorescence probes are used to undertake a systematic study to characterize the properties of the bilayer and the effects in the physical and chemical interventions in order to correlate them with changes in the ATPase activity. Using NaK-ATPase rich membrane prepration from pig kidney, the effects of incorporation of fluidizing agent (A,C) and n-octanol were studied at different depths of the bilayers by time resolved fluorescence measurements of DPH derivates.